Project Summary/Abstract Cigarette smoking is responsible for nearly one-half million deaths/yr. in the U.S. where direct healthcare costs to treat smoking and smoking-attributable illness exceed $170 billion/year. Only ~20-25% smokers achieve long- term abstinence using standard of care pharmacotherapies leaving the majority still smoking and seeking alternatives. The focus of this project is to further assess the protein properties of a novel nicotine-degrading enzyme and its suitability for use as a smoking cessation treatment. Enzymatic degradation of nicotine is a new approach for treating smoking cessation. Clinical ?proof-of-principle? has already been established for in vivo enzymatic degradation of drugs of abuse: two cocaine-degrading enzymes have demonstrated encouraging phase 1/2 results. Engineering of the biological properties of this enzyme is needed to reduce potential immunogenicity and to extend serum half-life, in order to ensure patient safety, sufficient stability and duration of enzymatic action. To this end we will investigate the effect of PEGylation, the chemical conjugation of large non-toxic polyethylene glycol (PEG) moieties to the surface of the enzyme, which provide steric masking of potential epitopes and enhanced hydrodynamic radius of the protein, decreasing its rate of renal clearance. PEGylated lead candidates will be progressed through a series of in vitro enzyme and serum stability assays, as well as in vivo models to assess PEGylation?s effects on immunogenic potential and pharmacokinetic properties. The goal of this project is to reduce the immunogenic potential and increase the serum stability of this novel enzyme though optimization of various PEGylation strategies, while maintaining its potent nicotine-degrading activity. Achieving these goals will provide supportive pre-clinical evidence to conduct further lead optimization efforts and multiple-dose studies in animal behavioral models of addiction (Phase II proposal); thereby progressing this novel nicotine degrading enzyme approach for smoking cessation into pre-clinical development.